US5420105A - Polymeric carriers for non-covalent drug conjugation - Google Patents

Polymeric carriers for non-covalent drug conjugation Download PDF

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US5420105A
US5420105A US08/095,515 US9551593A US5420105A US 5420105 A US5420105 A US 5420105A US 9551593 A US9551593 A US 9551593A US 5420105 A US5420105 A US 5420105A
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drug
ligand
polymeric carrier
binding
conjugate
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Linda M. Gustavson
David C. Anderson
Alton C. Morgan, Jr.
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Aletheon Pharmaceuticals Inc
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Poniard Pharmaceuticals Inc
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Priority claimed from US07/248,456 external-priority patent/US5252713A/en
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Priority to US08/095,515 priority Critical patent/US5420105A/en
Assigned to NEORX CORPORATION reassignment NEORX CORPORATION ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: MORGAN, ALTON CHARLES JR., ANDERSON, DAVID C., GUSTAVSON, LINDA M.
Priority to PCT/US1994/007734 priority patent/WO1995003064A1/fr
Priority to CA002167574A priority patent/CA2167574A1/fr
Priority to EP94923410A priority patent/EP0713395A4/fr
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    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6891—Pre-targeting systems involving an antibody for targeting specific cells
    • A61K47/6897—Pre-targeting systems with two or three steps using antibody conjugates; Ligand-antiligand therapies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6883—Polymer-drug antibody conjugates, e.g. mitomycin-dextran-Ab; DNA-polylysine-antibody complex or conjugate used for therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6891—Pre-targeting systems involving an antibody for targeting specific cells
    • A61K47/6897—Pre-targeting systems with two or three steps using antibody conjugates; Ligand-antiligand therapies
    • A61K47/6898—Pre-targeting systems with two or three steps using antibody conjugates; Ligand-antiligand therapies using avidin- or biotin-conjugated antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/36—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Actinomyces; from Streptomyces (G)
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/465—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from birds
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/473—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used alpha-Glycoproteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/721—Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to compositions and methods for making polymeric carriers for non-covalent binding of drugs.
  • the polymeric carriers are capable of binding one or multiple drug molecules.
  • the polymeric carriers are then covalently attached to a targeting protein, such as an antibody molecule, thereby forming an antibody/polymeric carrier/drug conjugate for targeting to defined populations of cells, such as cancer cells.
  • chemotherapeutic drugs to targeting molecules such as antibodies for the following reasons:
  • Pleiotropic drug resistance may arise following treatment with one of a number of chemotherapeutic drugs, resulting in inducing resistance to drugs of several classes.
  • the mechanism(s) of this resistance are not entirely known, but it is known that this resistance can be partially overcome by antibody targeting of drugs.
  • the therapeutic agent may be directly linked to the antibody through nucleophilic substitution of certain groups on the antibody (e.g., amino, carboxyl, or sulfhydryl) or the drug may be conjugated to the antibody via a hetero- or homobifunctional cross-linker.
  • certain groups on the antibody e.g., amino, carboxyl, or sulfhydryl
  • the drug may be conjugated to the antibody via a hetero- or homobifunctional cross-linker.
  • the linking group generally is heterobifunctional, having two different functionalities, one of which reacts with the drug and the other with the antibody.
  • Linking groups may be small or quite long.
  • a relatively small linking group is carbonyl diimidazole.
  • Large proteins or polymers (“carriers") have also been used as linking groups and offer the advantage of being able to bind many drug molecules to a single antibody molecule. Examples of -large proteins or polymers are poly-L-lysine, polyglutamate, dextran, and albumin, all of which have molecular weights in excess of 5000 daltons. These carriers generally are derivatized with small linking groups to bind drugs. See, for example, U.S. Pat. Nos. 4,699,784 and 4,046,722.
  • Drug conjugation to a protein or an antibody targeting molecule has generally been through covalent binding of the drug to the antibody directly or by covalently binding the drug molecule to the linking group.
  • a carrier molecule such as albumin or dextran
  • the drug undergoes a modification to allow for the covalent conjugation of the drug.
  • the drug modification often results in the loss of some of the activity of the drug molecule due to chemical modifications of some of the functional groups within the drug molecule.
  • the derivatization may not be completely specific for groups intended for linkage but may also modify groups important for drug activity.
  • Pretargeting approaches may be used to achieve therapeutic or diagnostic goals.
  • Pretargeting approaches involve the interaction of two members of a high affinity binding pair such as a ligand-anti-ligand binding pair.
  • Pretargeting is characterized by an uncoupling of the kinetics of the targeting moiety capable of localizing to a target site in vivo and the diagnostic or therapeutic active agent. This decoupling of the relatively slow localization kinetics of targeting moiety localization from the faster clearing active agent affords lower non-target exposure.
  • the present invention provides polymeric carriers comprising at least one drug-binding domain derived from a protein, wherein each drug-binding domain can non-covalently bind a drug.
  • the polymeric carrier preferably comprises multiple drug-binding domains, wherein the domains may be the same or different and therefore may bind the same or different drugs.
  • the polymeric carrier may be attached to a targeting protein, such as an antibody, that binds to a desired target site in vivo.
  • a targeting protein such as an antibody
  • the present invention thus provides targeting protein/polymeric carrier/drug conjugates comprising a targeting protein covalently bound to a polymeric carrier, wherein said polymeric carrier comprises one or more drug-binding domains having a drug non-covalently bound thereto, wherein each of said domains is derived from a drug-binding protein.
  • the targeting protein may be covalently bonded to the polymeric carrier directly or through a linker molecule.
  • Pharmaceutical preparations comprising such a conjugate in an aqueous solution (for in vivo administration for therapeutic purposes) also are disclosed.
  • the polymeric carrier may also be attached to a member of a high affinity binding pair, e.g., a ligand-anti-ligand binding pair as discussed herein.
  • a ligand-anti-ligand binding pair as discussed herein.
  • Such polymeric carrier-containing compounds may include one or more ligand or anti-ligand molecules.
  • the binding pair member (anti-ligand) serves to target a subsequently administered complementary binding pair member (ligand)-active agent conjugate to target sites characterized by previously localized targeting moiety-binding pair member (anti-ligand).
  • the present invention thus provides a binding pair member/polymeric carrier/drug conjugate comprising a ligand or anti-ligand covalently bound to a polymeric carrier, wherein said polymeric carrier comprises one or more drug-binding domains having a drug non-covalently bound thereto, wherein each of said domains is derived from a drug-binding protein.
  • ligand or anti-ligand molecules may be covalently bonded to the polymeric carrier directly or through a linker molecule.
  • An example of a ligand is biotin, with the complementary anti-ligand thereof being avidin or streptavidin, wherein biotin and avidin or streptavidin together form a ligand-anti-ligand binding pair.
  • Pharmaceutical preparations comprising such a conjugate in an aqueous solution (for in vivo administration for therapeutic purposes) also are disclosed.
  • the present invention also provides methods for producing polymeric carriers.
  • the carriers are derived from relatively large molecular weight proteins, and may be produced by such methods as peptide synthesis or recombinant DNA technology.
  • a method for preserving the therapeutic activity of a drug also is disclosed, said method comprising non-covalently binding the drug to a polymeric carrier.
  • the drug activity is thus preserved during subsequent chemical reactions, such as the reactions used to attach the polymeric carrier to a targeting protein to form a conjugate.
  • Drug activity also is preserved in vivo after administration of the conjugate to a human or mammalian host.
  • FIG. 1 depicts schematically a three-step protocol wherein targeting moiety-ligand is administered and permitted to localize to target.
  • Polymeric Carrier By the term “polymeric carrier” is meant a polymer, such as a polypeptide, comprising one or more drug-binding domains wherein the domains are capable of binding a drug through non-covalent bonds.
  • the polymeric carriers of the present invention are not naturally occurring, but are derived from naturally occurring proteins.
  • a polymeric carrier may comprise one or multiple drug-binding domains fabricated through such methods as peptide synthesis or recombinant DNA technology. The domains may then be polymerized to produce a multi-domain polymeric carrier.
  • a polymeric carrier of the present invention is able to non-covalently bind at least one drug through one or more non-covalent interactions or reversible interactions.
  • Covalent Bond A "covalent bond” is defined as the formation of a sigma bond between two organic molecules.
  • Non-covalent Bond A "non-covalent bond” is meant to include all interactions other than a covalent bond. Non-covalent bonds include ionic interactions, hydrogen bonding, pi-pi bonding, hydrophobic interactions, and van der Waals interactions.
  • Targeting moiety A molecule that binds to a defined population of cells.
  • the targeting moiety may bind a receptor, an oligonucleotide, an enzymatic substrate, an antigenic determinant, or other binding site present on or in the target cell population.
  • Targeting moieties that are proteins are referred to herein as "targeting proteins.”
  • Antibody is used throughout the specification as a prototypical example of a targeting moiety and a targeting protein. Tumor is used as a prototypical example of a target in describing the present invention.
  • Ligand/anti-ligand Pair A complementary/anti-complementary set of molecules that demonstrate specific binding, generally of relatively high affinity.
  • Exemplary ligand/anti-ligand pairs include zinc finger protein/dsDNA fragment, hapten/antibody, lectin/carbohydrate, ligand/receptor, and biotin/avidin. Biotin/avidin is used throughout the specification as a prototypical example of a ligand/anti-ligand pair.
  • Anti-ligand As defined herein, an "anti-ligand” demonstrates high affinity, and preferably, multivalent binding of the complementary ligand. Preferably, the anti-ligand is large enough to avoid rapid renal clearance, and contains sufficient multivalency to accomplish crosslinking and aggregation of targeting moiety-ligand conjugates. Univalent anti-ligands are also contemplated by the present invention. Anti-ligands of the present invention may exhibit or be derivatized to exhibit structural features that direct the uptake thereof, e.g., galactose residues that direct liver uptake. Avidin and streptavidin are used herein as prototypical anti-ligands.
  • Avidin and Streptavidin As defined herein, both of the terms “avidin” and “streptavidin” include avidin, streptavidin and derivatives and analogs thereof that are capable of high affinity, multivalent or univalent binding of biotin.
  • Ligand As defined herein, a "ligand” is a relatively small, soluble molecule that exhibits rapid serum, blood and/or whole body clearance when administered intravenously in an animal or human. Biotin is used as the prototypical ligand.
  • Pretargeting involves target site localization of a targeting moiety that is conjugated with one member of a ligand/anti-ligand pair; after a time period sufficient for optimal target-to-non-target accumulation of this targeting moiety conjugate, active agent conjugated to the opposite member of the ligand/anti-ligand pair is administered and is bound (directly or indirectly) to the targeting moiety conjugate at the target site (two-step pretargeting). Three-step and other related methods described herein are also encompassed.
  • the present invention addresses the problem of loss of drug activity due to attachment of drugs to various carriers through covalent bonds.
  • the present invention relates to a polymeric carrier containing one or multiple drug-binding domains (wherein each drug-binding domain non-covalently binds a drug), conjugates comprising a polymeric carrier bound to a targeting protein, ligand or anti-ligand, and conjugates comprising one or more drug molecules non-covalently bound to a polymeric carrier which in turn is bound to a targeting protein, ligand or anti-ligand.
  • the targeting protein is a protein that binds to a desired target site in vivo, thereby delivering the conjugate to the target site.
  • Targeting proteins include antibodies as well as proteinaceous ligands or anti-ligands, and are described in more detail below.
  • Ligands suitable for use within the present invention include biotin, haptens, lectins, epitopes, dsDNA fragments and analogs and derivatives thereof.
  • Useful complementary anti-ligands include avidin (for biotin), carbohydrates (for lectins), antibody, fragments or analogs thereof, including mimetics (for haptens and epitopes) and zinc finger proteins (for dsDNA fragments).
  • Preferred ligands and anti-ligands bind to each other with an affinity of at least about k D ⁇ 10 -9 M.
  • conjugates comprising a polymeric carrier as a drug active site protecting group.
  • the polymeric carrier serves to protect the drug's active functional groups during the chemical reactions used to attach the polymeric carrier to a targeting protein.
  • the polymeric carrier also protects the drug after in vivo administration of a drug(s)/polymeric carrier/targeting protein conjugate and minimizes nonspecific interactions of the drug moiety of the conjugate with cellular membranes.
  • Polymeric carriers are polymers such as polypeptides comprising one or a plurality of drug binding domains, which may be produced by such methods as peptide synthesis procedures or through cloning and expression of specific nucleotide sequences.
  • the polymeric carrier preferably contains multiple drug-binding domains, wherein the drug-binding domain may be derived from a large molecular weight polymer such as a protein and then polymerized.
  • the large protein can typically bind non-covalently only one or a few drug molecules.
  • the polymeric carrier polypeptides may be synthesized as a single polypeptide chain or as disulfide-bonded peptide chains.
  • the present invention provides a method for producing polymeric carriers. These carriers are prepared by first identifying a protein, generally a large molecular weight protein, that is able to non-covalently bind a particular drug of interest. A drug-binding domain is then isolated from the protein, wherein the drug-binding domain is capable of binding a drug of interest through non-covalent means.
  • large molecular weight proteins that can non-covalently bind to certain drug molecules include, but are not limited to, riboflavin-binding protein (RBP) to anthracyclines; albumin to certain lipophilic drugs such as anthracyclines, methotrexate, and cis-platinum; or one of the other proteins described below (e.g., in Table I).
  • RBP riboflavin-binding protein
  • albumin to certain lipophilic drugs such as anthracyclines, methotrexate, and cis-platinum
  • one of the other proteins described below e.g., in Table I.
  • the polymeric carriers may be produced through a variety of techniques. Such techniques include peptide synthesis to produce multiple copies of the domain, which may be joined to form a multi-domain polymeric carrier. Alternatively, single or multiple domain polymeric carriers may be produced through recombinant DNA technology.
  • Another aspect of the invention is a pharmaceutical composition which includes a conjugate comprising one or more drug molecules bound to a single- or multiple-domain polymeric carrier for prolonged serum half-life and increased efficacy.
  • These slow-release pharmaceutical compositions may include a conjugate comprising a drug non-covalently bound to a polymeric carrier which in turn may be attached to a targeting protein, ligand or anti-ligand.
  • conjugates are formed by covalently conjugating the polymeric carrier to ligand or anti-ligand, for example, wherein this conjugation is followed by non-covalent attachment of drug to the polymeric carrier-ligand or -anti-ligand compound.
  • the conjugate may comprise a polymeric carrier bound to a targeting protein, ligand or anti-ligand wherein the drug is to be added and non-covalently bound later, before use.
  • the non-covalent binding of the drug in the conjugates of the present invention permits slow release of the drug from the polymeric carrier in vivo. "Slow release” means that the serum half life of the drug is increased compared to free drug. The patient's tissues are exposed to the drug for a longer period of time than when free (i.e., unconjugated) drug is administered, and therapeutic efficacy thus is enhanced.
  • a drug-binding domain from a large molecular weight protein to form the conjugates of the invention because attaching a high molecular weight protein to a targeting protein may have an adverse effect on the desired biological activity (e.g., the "targeting" ability) of the targeting protein.
  • attaching the large protein to an antibody may impair the immunoreactivity and accessibility to tumors of the resulting immunoconjugate. This is especially true if more than one high molecular weight protein molecule is attached to a targeting protein molecule.
  • RBP is a 50-kilodalton glycoprotein that binds one mole of drug per mole of protein.
  • the total molecular weight of the multi-domain polymeric carrier preferably is less than about 60,000 daltons.
  • Non-covalent binding of the drug preserves the activity of the drug, as discussed above.
  • a polymeric carrier also serves to protect the active functional groups on the drug molecule by non-covalently binding to the drug molecule.
  • the enveloping of the drug by the polymeric carrier serves to protect the functional groups of the drug molecule from any subsequent derivatization conditions (used to conjugate the carrier to the targeting protein, ligand or anti-ligand) and to block nonspecific interactions between the drug functional groups and non-target cell surfaces during in vivo administration of the targeting protein, ligand or anti-ligand conjugate.
  • the process of isolating a polymeric carrier from a drug-binding, large molecular weight protein begins with the identification of a large protein that can non-covalently bind the drug of interest. Examples of such protein/drug pairs are shown in Table I.
  • the drugs in the Table are anti-cancer drugs.
  • Other drug-binding proteins may be identified by appropriate analytical procedures, including Western blotting of large proteins or protein fragments and subsequent incubation with a detectable form of drug.
  • Alternative procedures include combining a drug and a protein in a solution, followed by size exclusion HPLC gel filtration, thin-layer chromatography (TLC), or other analytical procedures that can discriminate between free and protein bound drug.
  • Detection of drug binding can be accomplished by using radiolabeled, fluorescent, or colored drugs and appropriate detection methods. Equilibrium dialysis with labeled drug may be used.
  • Alternative methods include monitoring the fluorescence change that occurs upon binding of certain drugs (e.g., anthracyclines or analogs thereof, which should be fluorescent).
  • drug and protein are mixed, and an aliquot of this solution (not exceeding 5% of the column volume of an HPLC column, such as a Biosil TSK-250 7.5 ⁇ 30 cm column) is loaded onto the HPLC column.
  • the flow rate is 1 ml/min.
  • the drug bound to protein will elute first, in a separate peak, followed by free drug, eluting at a position characteristic of its molecular weight. If the drug is doxorubicin, both a 280-nm as well as a 495-nm adsorptive peak will correspond to the elution position of the protein if interaction occurs. The elution peaks for other drugs will indicate whether drug binding occurs.
  • the drug-binding domain is identified and isolated from the protein by any suitable means. Protein domains are portions of proteins having a particular function or activity (in this case, non-covalent binding of drug molecules).
  • the present invention provides a process for producing a polymeric carrier, comprising the steps of generating peptide fragments of a protein that is capable of non-covalently binding a drug and identifying a drug-binding peptide fragment, which is a peptide fragment containing a drug-binding domain capable of non-covalently binding the drug, for use as the polymeric carrier.
  • One method for identifying the drug-binding domain begins with digesting or partially digesting the protein with a proteolytic enzyme or specific chemicals to produce peptide fragments.
  • useful proteolytic enzymes include lys-C-endoprotease, arg-C-endoprotease, V8 protease, endoprolidase, trypsin, and chymotrypsin.
  • chemicals used for protein digestion include cyanogen bromide (cleaves at methionine residues), hydroxylamine (cleaves the Asn-Gly bond), dilute acetic acid (cleaves the Asp-Pro bond), and iodosobenzoic acid (cleaves at the tryptophane residue). In some cases, better results may be achieved by denaturing the protein (to unfold it), either before or after fragmentation.
  • the fragments may be separated by such procedures as high pressure liquid chromatography (HPLC) or gel electrophoresis.
  • HPLC high pressure liquid chromatography
  • gel electrophoresis The smallest peptide fragment capable of drug binding is identified using a suitable drug-binding analysis procedure, such as one of those described above.
  • One such procedure involves SDS-PAGE gel electrophoresis to separate protein fragments, followed by Western blotting on nitrocellulose, and incubation with a colored drug like adriamycin. The fragments that have bound the drug will appear red. Scans at 495 nm with a laser densitometer may then be used to analyze (quantify) the level of drug binding.
  • the smallest peptide fragment capable of non-covalent drug binding is used. It may occasionally be advisable, however, to use a larger fragment, such as when the smallest fragment has only a low-affinity drug binding domain.
  • the amino acid sequence of the peptide fragment containing the drug-binding domain is elucidated.
  • the purified fragment containing the drug-binding region is denatured in 6M guanidine hydrochloride, reduced and carboxymethylated by the method of Crestfield et al., J. Biol. Chem. 238:622, 1963.
  • As little as 20 to 50 picomoles of each peptide fragment can be analyzed by automated Edman degradation using a gas-phase or liquid-pulsed protein sequencer (commercially available from Applied Biosystems, Inc.). If the peptide fragment is longer than 30 amino acids, it will most likely have to be fragmented as above and the amino acid sequence patched together from sequences of overlapping fragments.
  • Peptide amides can be made using 4-methylbenzhydrylamine-derivatized, cross-linked polystyrene- 1% divinylbenzene resin and peptide acids made using PAM (phenylacetamidomethyl) resin (Stewart et al., "Solid Phase Peptide Synthesis," Pierce Chemical Company, Rockford, Ill., 1984).
  • the synthesis can be accomplished either using a commercially available synthesizer, such as the Applied Biosystems 430A, or manually using the procedure of Merrifield et al., Biochemistry 21:5020-31, 1982; or Houghten, PNAS 82:5131-35, 1985.
  • polymeric carriers can be tested for drug binding using size-exclusion HPLC, as described above, or any of the other analytical methods listed above.
  • the second synthetic mechanism involves the determination of a DNA sequence which will encode the desired amino acid sequence (i.e., the amino acid sequence of the drug-binding peptide fragment: determined above).
  • a DNA sequence may be determined because the genetic code (i.e., the three-base sequence or codon in an mRNA which specifies a given amino acid) is known.
  • a DNA sequence which encodes the polymeric carrier may be synthesized in in vitro by standard oligonucleotide synthesis procedures. See, for example, U.S. Pat. Nos. 4,500,707 and 4,668,777.
  • the synthetic DNA fragment encoding the polymeric carrier is cloned and expressed using recombinant DNA technology.
  • Microorganisms which have been used as host cells include, but are not limited to, prokaryotes, such as gram-negative and gram-positive bacteria, and eukaryotes, such as yeast or mammalian cell lines.
  • the DNA sequence is inserted in vitro into a vector capable of replication in certain host microorganisms.
  • the vector typically is derived from a plasmid or a virus.
  • a number of cloning vector/host cell systems have been developed including vectors suitable for transforming the gram-negative bacterium E. coli (Old and Primrose, Principals Of Gene Manipulation, 2d ed., Univ. of California Press, 1981, pp. 32-35 and 46-47), gram-positive bacteria Bacillus subtilis (Old and Primrose, pp. 51-53), or eukaryotic microorganisms such as yeast (Old and Primrose, pp. 62-68) "Shuttle vectors," which may be transferred (along with the cDNA they carry) between the host microorganisms, E. coli and yeast, have been described by Storms et al., J.
  • An appropriate microorganism strain is transformed with the recombinant expression vector, then cultured in a suitable growth medium under conditions appropriate for production of the desired polypeptide.
  • Expression vector systems may be engineered so that expression of the foreign protein may be regulated by chemical or temperature induction. Proteins which are secreted out of the host cells may be isolated from the growth media by conventional protein purification procedures.
  • the cells are harvested and then lysed through procedures which may be mechanical (e.g., sonication, homogenization, freeze-thawing, nitrogen compression-decompression, etc.), chemical (e.g., treatment with detergents such as sodium dodecyl sulfate, guanidine HCl or NP-40), enzymatic (such as by using lysozyme), or combinations thereof.
  • the desired polypeptide is then purified from the lysate using conventional procedures.
  • polypeptides produced by cloning and expressing DNA sequences encoding one or more drug-binding domains are used as polymeric carriers.
  • the recombinant cells may be cultured to produce large quantities of the polymeric carrier polypeptides, and these carriers may be attached to various targeting proteins to form conjugates capable of non-covalently binding drug molecules.
  • Another process for isolating a polymeric carrier involves cloning the gene which encodes a large molecular weight protein that can non-covalently bind to a drug of interest. Procedures for isolating and cloning DNA sequences which encode such proteins are known. See, for example, Lawn et al., Nucleic Acids Research 9:6103-14, 1981, in which isolation of cDNA which encodes the human serum albumin (HSA) protein is described.
  • HSA human serum albumin
  • a cloned gene encoding a drug-binding protein may be isolated from a recombinant microorganism and fragmented using restriction endonucleases. The resulting gene fragments are subcloned and expressed in a suitable host/vector system, thereby producing fragments of the drug-binding protein. The peptide fragments produced by the various recombinant microorganisms transformed with the subcloned DNA are analyzed for drug-binding ability. Recombinant cultures producing peptide fragments comprising the drug-binding domain thus are identified.
  • the polymeric carrier preferably comprises more than one drug-binding domain. Conjugates of such polymeric carriers and targeting proteins, ligands or anti-ligands may be used to deliver multiple drug molecules to target cells, thus enhancing the therapeutic effect against the target cells.
  • polymeric carriers comprising more than one drug-binding domain may be derived from the peptides containing single domains which are produced by any of the above-described methods.
  • Several drug-binding domains can be covalently joined together, after refolding, using bifunctional linkers to form polymeric carriers.
  • the linkers are selected to give optimal polymerization and generally consist of variable-length spacer groups with a chemically reactive group at each end.
  • the two chemically reactive groups may be the same or different, and each will react with a functional group on a peptide fragment, thereby joining peptide fragments together through the linker.
  • a linker may comprise are amine-reactive groups such as esters and sulfhydryl-reactive groups such as maleimides.
  • the spacer portion of the linker preferably is large enough to reduce steric hindrance during reaction with the peptide fragments, yet small enough so that the linker molecules used to form a multi-domain polymeric carrier do not significantly increase the molecular weight of the carrier.
  • the spacer may, for example, comprise a chain of from two to four methylene groups or a single cyclohexane ring.
  • the total size of the polymeric carrier will vary according to such factors as the therapeutic activity of the particular drug to be used (e.g., whether attachment of multiple drug molecules to the carrier is desirable), the susceptibility of a particular targeting protein, ligand or anti-ligand to loss of targeting ability when a high molecular weight polypeptide (i.e., the carrier) is attached thereto, and the size of the drug-binding peptide(s) from which the polymeric carrier is formed.
  • a polymeric carrier comprises from two to twenty, preferably from two to about ten, drug-binding domains.
  • the polymeric carrier preferably has a molecular weight of about 35 kilodaltons or less.
  • the choice of a particular polypeptide for use as a polymeric carrier may be influenced by several factors. Stability of the non-covalent drug binding to the drug-binding domain is one such factor.
  • the water solubility of the polymeric carrier component of the conjugate is considered.
  • the polymeric carrier may comprise amino acid sequences extraneous to the actual drug-binding domain. Certain amino acid residues may be added at the termini of the domain-containing peptide, wherein the amino acid residues comprise chemically reactive groups that will react with one of the above-described bifunctional cross-linkers.
  • amino acid sequences may be added to the drug-binding domain in order to achieve the secondary structure required for a particular desirable biological property.
  • Various amino acid sequences may be added during peptide synthesis or DNA synthesis to the peptide fragment originally derived from the drug binding protein. These amino acid sequences may be chosen to increase the water solubility of the resulting polymeric carrier, for example. Such sequences may be chosen based on knowledge of the sequences that confer hydrophilicity on other known proteins, for example.
  • conjugates of the present invention may be desirable to prepare conjugates of the present invention that comprise more than one type of drug. This is especially advantageous when two or more drugs have a synergistic therapeutic effect on the target cells.
  • Administration of more than one type of drug is desirable in the treatment of certain diseases such as cancer, especially in view of the heterogeneous cell populations found within some tumors.
  • a polymeric carrier of the present invention may comprise two or more different types of drug-binding domains.
  • the domains are each isolated from different large molecular weight proteins, then are joined to form a polymeric carrier, using the procedures described above.
  • a particular drug-binding domain may be capable of binding more than one type of drug.
  • Polymeric carriers comprising multiple copies of such a domain may be incubated with the different drugs to bind two or more different types of drugs to the polymeric carrier.
  • Any suitable procedure may be used for non-covalently binding a drug of interest to the polymeric carrier.
  • an excess of the drug is incubated with the carrier in a buffered aqueous solution to bind the drug to the carrier.
  • a polymeric carrier of the present invention having one or more drug molecules bound thereto, is administered to a human or mammalian host for therapeutic purposes.
  • These polymeric carriers are useful as slow-release drug delivery systems.
  • the polymeric carriers produced by the above-described procedures may be attached to targeting proteins, ligands or anti-ligands or other targeting moieties.
  • the targeting protein serves to deliver the conjugate to a specific cellular or tissue target site when administered in vivo.
  • the targeting is preferably accomplished by immune selectivity through antigen/antibody interactions.
  • targeting moiety binds to a defined target cell population, such as tumor cells.
  • Preferred targeting moieties useful in this regard include antibody and antibody fragments, proteinaceous ligands or anti-ligands, non-proteinaceous ligands or anti-ligands, peptides, and hormones. Proteins corresponding to or binding to known cell surface receptors (including low density lipoproteins, transferrin and insulin), fibrinolytic enzymes, anti-HER2, platelet binding proteins such as annexins, and biological response modifiers (including interleukin, interferon, erythropoietin and colony-stimulating factor) are also preferred targeting moieties.
  • anti-EGF receptor antibodies which internalize following binding to the EGF receptor and which traffic to the nucleus, are preferred targeting moieties for use in the present invention to facilitate delivery of Auger emitters and nucleus binding drugs to target cell nuclei.
  • Oligonucleotides e.g., antisense oligonucleotides that are complementary to portions of target cell nucleic acids (DNA or RNA), are also useful as targeting moieties in the practice of the present invention. Oligonucleotides binding to cell surfaces are also useful. Analogs of the above-listed targeting moieties that retain the capacity to bind to a defined target cell population may also be used within the claimed invention.
  • synthetic targeting moieties may be designed.
  • targeting moieties of the present invention are also useful as targeting moieties of the present invention.
  • One targeting moiety functional equivalent is a "mimetic" compound, an organic chemical construct designed to mimic proper configuration and/or orientation for targeting moiety-target cell binding.
  • Another targeting moiety functional equivalent is a short polypeptide designated as a "minimal” polypeptide, constructed using computer-assisted molecular modeling and mutants having altered binding affinity, which minimal polypeptides exhibit the binding affinity of the targeting moiety.
  • Proteinaceous targeting moieties of the present invention are referred to as "targeting proteins.”
  • Suitable targeting proteins include, but are not limited to, antibodies and antibody fragments; serum proteins; enzymes; peptide hormones; and biologic response modifiers.
  • suitable biologic response modifiers which may be used are lymphokines such as interleukins (e.g., IL-1, -2, -3, -4, -5, and -6) or interferons (e.g., alpha, beta, and gamma interferon), erythropoietin, and colony stimulating factors (e.g., G-CSF, GM-CSF, and M-CSF).
  • Peptide hormones include melanocyte stimulating hormone, follicle stimulating hormone, luteinizing hormone, and human growth hormone.
  • Enzymes include fibrinolytic enzymes such as tissue-type plasminogen activator, streptokinase, and urokinase.
  • Serum proteins include human serum albumin.
  • proteins may be modified; e.g., to produce variants and fragments of the proteins, as long as the desired biological property (i.e., the ability to bind to the target site) is retained.
  • the proteins may be modified by using various genetic engineering or protein engineering techniques. Another type of modification involves chemically modifying targeting proteins to effect a shift in the isoelectric point of the resulting "charge modified" protein, as described in co-pending U.S. patent application Ser. No. 157,273, filed on Feb. 17, 1988 and now U.S. Pat. No. 5,322,678, entitled "Alteration of Pharmacokinetics of Proteins by Charge Modification".
  • the serum half-life, biodistribution, immunogenicity, and other properties of targeting proteins may be altered by modifying the charge of the protein.
  • the antibodies employed as targeting proteins in the present invention may be intact antibody molecules, fragments thereof, or functional equivalents thereof, including genetically engineered variations thereof.
  • antibody fragments are F(ab') 2 , Fab', Fab, and Fv fragments, which may be produced by conventional procedures or by genetic or protein engineering.
  • polyclonal antibodies may be employed in the present invention, monoclonal antibodies (MAbs) are preferred.
  • MAbs monoclonal antibodies that bind to a specific type of cell have been developed, including MAbs specific for tumor-associated antigens in humans.
  • MAbs that may be used are anti-TAC or other interleukin-2 receptor antibodies, 9.2.27 and NR-ML-05 to the 250-kilodalton human melanoma-associated proteoglycan; NR-LU-10 to the 37 to 40-kilodalton pancarcinoma glycoprotein; and OVB3 to an as yet unidentified tumor-associated antigen.
  • Human monoclonal antibodies or "humanized” murine antibodies are also useful as targeting moieties in accordance with the present invention.
  • murine monoclonal antibody may be "humanized” by genetically recombining the nucleotide sequence encoding the murine Fv region (i.e., containing the antigen binding site which antibodies are also known as chimeric antibodies) or the complementarity determining regions thereof with the nucleotide sequence encoding at least a human constant domain region and an Fc region, e.g., in a manner similar to that disclosed in European Patent Application No. 0,411,893 A2. Some additional murine residues may also be retained within the human variable region framework domains to ensure proper target site binding characteristics.
  • Humanized targeting moieties are recognized to decrease the immunoreactivity of the antibody or polypeptide in the host recipient, permitting an increase in the half-life and a reduction in the possibility of adverse immune reactions.
  • the polymeric carriers of the present invention may be attached to ligands or anti-ligands to form drug-polymeric carrier-ligand or -anti-ligand conjugates having diagnostic or therapeutic use.
  • Such polymeric carriers include or are derivatized to include a ligand or anti-ligand conjugation group to facilitate attachment of the compound to a ligand or anti-ligand.
  • a ligand or anti-ligand conjugation group is a chemically reactive functional group that will react with a ligand or anti-ligand under conditions that do not adversely affect the ligand or anti-ligand, including the capacity of the ligand or anti-ligand to bind to its complementary binding pair member.
  • Ligand or anti-ligand conjugation groups therefore are sufficiently reactive with a functional group on a ligand or anti-ligand so that the reaction can be conducted under relatively mild reaction conditions including those described above for protein-chelate conjugation.
  • protein conjugation groups may correspond to ligand or anti-ligand conjugation groups.
  • Suitable ligand or anti-ligand conjugation groups therefore include, but are not limited to, active esters, isothiocyanates, amines, hydrazines, thiols, and maleimides.
  • active esters are thiophenyl ester, 2,3,5,6-tetrafluorophenyl ester, and 2,3,5,6-tetrafluorothiophenyl ester.
  • the preferred active esters may comprise a group that enhances water solubility, at the para (i.e., 4) position on the phenyl ring. Examples of such groups are CO 2 H, SO 3 - , PO 3 2- , OPO 3 2- , and O(CH 2 CH 2 O) n CH 3 groups.
  • suitable conjugations groups are those functional groups that react with a ligand or anti-ligand functional group (e.g., a terminal carboxy group) or a functional group which the ligand or anti-ligand has been derivatized to contain (e.g., an alcohol or an amine group produced by the reduction of a terminal carboxy moiety).
  • a ligand or anti-ligand functional group e.g., a terminal carboxy group
  • a functional group which the ligand or anti-ligand has been derivatized to contain e.g., an alcohol or an amine group produced by the reduction of a terminal carboxy moiety.
  • conjugation groups such as those recited above, that are capable of reacting with --COOH, --OH or --NH 2 groups are useful conjugation groups for producing biotin-containing conjugates of this aspect of the present invention.
  • Exemplary biotin-COOH conjugation groups are amines, hydrazines, alcohols and the like.
  • Exemplary biotin-OH conjugation groups are tosylates (Ts), active esters, halides and the like, with exemplary groups being reactive with biotin-O-Ts including amines, hydrazines, thiols and the like.
  • Exemplary biotin-NH 2 conjugation groups are active esters, acyl chlorides, tosylates, isothiocyanates and the like.
  • a variety of procedures may be used to attach the polymeric carrier to a targeting protein, such as an antibody and proteinaceous ligands or anti-ligands such as avidin or streptavidin.
  • a targeting protein such as an antibody and proteinaceous ligands or anti-ligands such as avidin or streptavidin.
  • Both the polymeric carrier and the targeting protein or proteinaceous ligand or anti-ligand, such as avidin or streptavidin are polypeptides which contain a variety of functional groups, e.g., carboxylic acid (COOH) or free amine (--NH 2 ) groups, which are available for reaction with a suitable functional group to covalently bind the polymeric carrier to the targeting protein.
  • COOH carboxylic acid
  • --NH 2 free amine
  • reaction with a water-soluble carbodiimide coupling reagent may be used to form bonds between a free amino group on one reactant species and a COOH group on the other reactant species.
  • the antibody, the proteinaceous ligand or anti-ligand and/or the polymeric carrier may be derivatized to expose or attach additional reactive functional groups.
  • the derivatization may involve attachment of any of a number of linker molecules, such as those available from Pierce Chemical Company, Rockford, Ill. (see Pierce 1988 General Catalog, pp. 221-250).
  • derivatization may involve chemical treatment of the antibody, ligand or anti-ligand, e.g., oxidative cleavage of vicinal hydroxyls on the sugar moiety of a glycoprotein antibody with periodate to generate free aldehyde groups.
  • the free aldehyde groups on the antibody, ligand or anti-ligand may be reacted with free amine groups on the polymeric carrier to form the desired bond. See U.S. Pat. No. 4,671,958. Procedures for generation of free sulfhydryl groups on antibodies or antibody fragments are also known. See U.S. Pat. No. 4,659,839. Many procedures and linker molecules for attachment of various compounds to proteins such as antibodies or proteinaceous ligands or anti-ligands are known. See, for example, U.S. Pat. Nos. 4,671,958; 4,414,148; 4,046,722; 4,699,784; and 4,680,338.
  • Biotin has a terminal carboxy moiety which may be reacted with a suitable ligand conjugation group, such as an amine or a hydroxyl in the presence of a coupling agent (e.g., DCC) or the like.
  • a suitable ligand conjugation group such as an amine or a hydroxyl in the presence of a coupling agent (e.g., DCC) or the like.
  • the terminal carboxy moiety may be derivatized to form an active ester, which is suitable for reaction with a suitable ligand conjugation group, such as an amine, a hydroxyl, another nucleophile, or the like.
  • the terminal carboxy moiety may be reduced to a hydroxy moiety for reaction with a suitable ligand conjugation group, such as a halide (e.g., iodide, bromide or chloride), tosylate, mesylate, other good leaving groups or the like.
  • a suitable ligand conjugation group such as a halide (e.g., iodide, bromide or chloride), tosylate, mesylate, other good leaving groups or the like.
  • the hydroxy moiety may be chemically modified to form an amine moiety, which may be reacted with a suitable ligand conjugation group, such as an active ester or the like.
  • a polymeric carrier/drug conjugate or a targeting protein, ligand or anti-ligand/polymeric carrier/drug conjugate of the present invention may be administered for therapeutic purposes to a human or mammalian host by any suitable means.
  • the conjugate may be administered intravenously, intraarterially, or peritoneally, for example, with the choice being determined by such factors as the location of the target site(s) within the body.
  • the dosage will vary according to such factors as the type of drug in the conjugate, the number of drug molecules attached to the polymeric carrier, and the specificity of the targeting protein or the binding affinity of the ligand-anti-ligand pair.
  • Elevated doses e.g., ranging from about 2 to about 10 times higher, can be used when pretargeting procedures are employed, because of the decoupling of targeting moiety localization and radionuclide localization.
  • a physician skilled in the field to which this invention pertains will be able to determine the proper dosage of a given conjugate.
  • pretargeting encompasses two protocols, termed the three-step and the two-step.
  • targeting moiety-ligand is administered and permitted to localize to target.
  • Targeting moiety-ligand conjugates may be prepared in accordance with known techniques therefor.
  • Anti-ligand is then administered to act as a clearing agent and to facilitate and direct the excretion of circulating targeting moiety-ligand.
  • the anti-ligand also binds to target-associated targeting moiety-ligand.
  • a conjugate employing a compound of the present invention is administered, having the following structure:
  • the drug-bearing ligand conjugate either binds to target-associated targeting moiety-ligand-anti-ligand or is rapidly excreted, with the excretion proceeding primarily through the renal pathway.
  • Some drug-bearing ligand conjugate may bind to residual circulating anti-ligand-containing conjugate; however, the protocol is designed to minimize such binding. Consequently, the target-non-target ratio of active agent is improved, and undesirable hepatobiliary excretion and intestinal uptake of the active agent are substantially decreased.
  • Two-step pretargeting involves administration of targeting moiety-anti-ligand, which may be prepared in accordance with known techniques therefor. After permitting the administered agent to localize to target, a ligand-polymer-drug of the present invention is administered.
  • a clearing agent is administered to remove circulating targeting moiety-anti-ligand without binding of clearing agent to target-associated targeting moiety-anti-ligand. In this manner, the target-non-target ratio of the active agent bearing ligand is increased, and undesirable hepatobiliary excretion and intestinal uptake of the active agent are substantially decreased.
  • a targeting moiety may be covalently linked to both ligand and a polymeric carrier bearing therapeutic agents and administered to a recipient. Subsequent administration of anti-ligand crosslinks targeting moiety-ligand-polymer/therapeutic agent conjugates bound at the surface, inducing internalization of the conjugate (and thus the active agent).
  • targeting moiety-ligand may be delivered to the target cell surface, followed by administration of anti-ligand-polymeric carrier-therapeutic agent(s).
  • the drug adriamycin i.e., doxorubicin
  • Riboflavin-binding protein is used as the source of the polymeric carrier because this compound is known to non-covalently bind adriamycin.
  • Other anthracycline drugs may be used in place of or in addition to adriamycin in the following procedures.
  • the sequence of CRBP is known (Protein Information Resource Data Bank, release 14 (1987)).
  • the sequence of CRBP contains 5 arginines and 7 methionines.
  • the proteolytic enzyme arg-C-endoprotease and the chemical peptide digestion agent cyanogen bromide are used initially to generate fragments for testing of drug (adriamycin) binding.
  • Other chemical cleavage methods or proteases could also be used.
  • the fragments are 56, 20, 7, 5, 37 and 92 amino acid residues long.
  • the amino acid fragments are 21, 122, 7, 4, 17, 5, 17 and 25 residues long.
  • the CRBP protein cystines are reduced for one day at 37° C. in 6 M guanidine hydrochloride at pH 8.5 in 0.1 M tris buffer with a 100-fold excess of dithiothreitol (DTT) to protein cysteines.
  • the cysteines may be carboxymethylated with a 5-fold excess of iodoacetic acid to DTT thiols for one hour at 37° C.
  • the protein is microdialyzed against water, or an appropriate buffer for digestion. For cyanogen bromide digestion, 200 ⁇ g protein is dissolved in 400 ⁇ l of 70% formic acid containing 100 moles of cyanogen bromide per mole methionine, and reacted in the dark, under nitrogen for 24 hours at 37° C.
  • the mixture is diluted 10-fold with water, lyophilized, run over a Vydac C-4 5 ⁇ 0.4 ⁇ 25 cm reversed-phase column. Elution is with a gradient of 1% per minute from water plus 0.1% trifluoroacetic acid (TFA) to 100% acetonitrile plus 0.1% TFA.
  • TFA trifluoroacetic acid
  • CRBP protein 200 ⁇ g reduced (or reduced and carboxymethylated) CRBP protein is dissolved in 0.1 M sodium bicarbonate, pH 8.0, and digested with 10 ⁇ g submaxillaris protease (an arg-C-endoprotease) for 14 hours.
  • the fragments are purified as above.
  • the purified protein fragments (obtained by either chemical or enzymatic digestion) are incubated in 0.1 M phosphate or 0.1 M hepes buffer at pH 7.0 for 1 hour with a 100-fold excess of adriamycin and then eluted over an appropriate gel-filtration column.
  • the peptide peak is checked spectro-photometrically for elution peaks at 280 nm and 495 nm to detect bound adriamycin. This procedure can be repeated with other peptide fragments or other digestion product fragments until a tight binding fragment (preferably Kd approximately less than or equal to 1 uM, as measured by equilibrium dialysis or a fluorescence or spectrophotometric titration) is discovered.
  • a tight binding fragment preferably Kd approximately less than or equal to 1 uM, as measured by equilibrium dialysis or a fluorescence or spectrophotometric titration
  • Smaller versions (i.e., subfragments) of the peptide fragment may be synthesized by solid-phase synthesis methodology, as mentioned above, to find the minimal size binding domain, which will tightly bind to the drug adriamycin.
  • peptides about 30-50 amino acid residues in length, overlapping by 15 residues can be synthesized using the solid-phase peptide synthesis methodology (described above) from the known CRBP sequence. These peptides can be tested for adriamycin binding as above.
  • the polymerization of the minimal length peptides retaining tight binding to adriamycin is achieved using bifunctional cross-linking reagents.
  • the choice of cross-linking reagent depends on the amino acid composition of the adriamycin-binding domain. If the adriamycin-binding domain contains 2 or more lysine residues, for example, polymerization may be achieved using amine-reactive, bifunctional cross-linking reagents, such as bis(sulfosuccimidyl) subcrate. Polymerization is achieved after binding of the drug binding domain to adriamycin. A 100-fold excess of the drug is incubated with the peptide in a buffered solution, as described above.
  • the polymeric carrier comprises less than 20 copies of the drug-binding domain.
  • the resulting polymerized polymeric carrier having adriamycin bound thereto can be purified as necessary by sizeexclusion HPLC.
  • the polymeric carrier bound to adriamycin is attached to the targeting protein by a similar cross-linking procedure.
  • the procedure may vary according to the type of targeting protein used.
  • One of the methods described above for attaching carriers to targeting proteins as well as proteinaceous or non-proteinaceous ligands or anti-ligands to form conjugates of the invention e.g., through the use of bifunctional cross-linkers, such as the heterobifunctional cross-linking agent succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) available from Pierce Chemical Company
  • SMCC heterobifunctional cross-linking agent
  • HSA Human serum albumin
  • coli HB101 cells are transformed with the resulting plasmids and cultured to produce the HSA protein fragments encoded by the HSA gene fragments.
  • These protein fragments are purified from samples of each culture by a standard procedure, such as immunoprecipitation, followed by SDS polyacrylamide gel electrophoresis. See Kessler, J. Immunology 117:1432-90, 1976; and Laemmli, Nature 277:680-85, 1970.
  • Each protein fragment is analyzed to determine its ability to bind a drug of interest by one of the procedures described above.
  • the drug adriamycin is combined with each protein fragment and the samples are each analyzed to detect free versus protein-bound drug.
  • One method of analysis involves subjecting the protein fragment samples (either purified or in the form of the E. coli cell lysates) to electrophoresis on an SDS-polyacrylamide gel. The fragments (separated according to molecular weight) are transferred from the gel to a nitrocellulose sheet in accordance with the known "Western blot" technique. A solution containing adriamycin is contacted with the nitrocellulose sheet.
  • the polypeptide bands which bind the drug will appear red, the color imparted by adriamycin.
  • the culture(s) found to produce a relatively small protein fragment with sufficient affinity for the drug are cultured to produce the polymeric carrier (i.e., the HSA protein fragment) on a larger scale.
  • the amino acid sequence of the peptide fragment may be determined, and the fragment may be produced by peptide synthesis procedures.
  • a polymeric carrier comprising multiple drug-binding domains
  • multiple copies of the peptide fragment produced above may be joined together using a bifunctional cross-linker.
  • a number of different cross-linkers may be used, depending on the amino acid sequence of the peptides to be joined.
  • the linker may be chosen from those described in the Pierce Chemical Company Catalog, as discussed above.
  • One cross-linking procedure is presented in Example 1.
  • An alternative method for producing the polymeric carrier involves determining the amino acid sequence of the peptide fragment, synthesizing a DNA sequence that encodes a polypeptide comprising at least one copy of the peptide fragment, expressing the DNA sequence in recombinant host cells (thereby producing the polypeptide), and purifying the polypeptide from the recombinant cells for use as a polymeric carrier.
  • the DNA sequence may encode a polymeric carrier comprising multiple drug-binding domains.
  • multiple copies of a single drug-binding peptide fragment produced by the recombinant cells may be purified and enzymatically ligated together to form multi-domain polymeric carriers.
  • a polymeric carrier such as one produced as described in Example 1 or 2 is covalently bound to an antibody as follows. This is an alternative procedure to the methods for forming conjugates described in Example 1.
  • the polymeric carrier is conjugated to a monoclonal antibody through a thioether linkage.
  • the polymeric carrier is first reacted with succinimidyl 4-(N-maleimido-methyl)cyclohexane-1-carboxylate (SMCC) at a molar ratio of 1:10 (carrier:linker).
  • SMCC succinimidyl 4-(N-maleimido-methyl)cyclohexane-1-carboxylate
  • Excess heterobifunctional linker reagent is removed from derivatized polymeric carrier by gel filtration.
  • the antibody is treated with 25 mM dithio-threitol (DTT) in 0.01 M phosphate-buffered saline (PBS), pH 7.5, and excess DTT is removed by gel filtration.
  • DTT dithio-threitol
  • PBS phosphate-buffered saline
  • PBS phosphate-buffered saline
  • conjugation reaction mixtures are then fractionated by FPLC gel filtration on a TSK 3000 column at 0.5 ml/min to separate the immunoconjugate from unconjugated antibody and unreacted derivatized carrier.
  • the resulting immunoconjugate is mixed with the drug in a buffered solution, whereby the drug becomes associated with the polymeric carrier.
  • Non-bound drug is removed by gel filtration or dialysis.
  • the thus-produced conjugate of the present invention may be administered to a patient bearing a target site to which the antibody binds, wherein the target site is to be treated with the drug.
  • the antibody may be a monoclonal antibody that binds to a tumor, and the drug is an anti-cancer drug.
  • HSA human serum albumin
  • the full-length protein human serum albumin (HSA) is purified from human blood plasma or from recombinant cells by known procedures. See, for example, U.S. Pat. No. 4,684,723; and Lawn et al., Nucleic Acids Research, vol. 9, No 22, 1981.
  • the protein is subjected to digestion with a proteolytic enzyme to generate polypeptide fragments which are separated by electrophoresis (e.g., on an SDS-polyacrylamide gel).
  • the ability of each polypeptide fragment to bind a particular drug of interest is analyzed by procedures which detect protein-bound drug versus free drug, such as those described in Examples 1 and 2.
  • the drug is a drug that the parent protein binds non-covalently (see Table I).
  • Drug-binding polypeptide fragments suitable for use as polymeric carriers thus are identified. If desired, smaller peptide fragments may be generated by enzymatic or chemical cleavage of the thus-identified drug-binding polypeptide fragment. The drug-binding assay is repeated on the smaller fragments to identify the smallest peptide fragment comprising a drug-binding domain.
  • the amino acid sequence of each carrier is determined using the standard Edman degradation process, as described above. Once the amino acid sequence is determined, the carriers are synthesized as needed using a commercially available peptide synthesizer. Multi-domain polymeric carriers may be produced by joining multiple copies of the peptide together using bifunctional linkers.
  • a DNA sequence which encodes the desired amino acid sequence (preferably multiple copies thereof) is synthesized in vitro.
  • the synthesized DNA sequence is inserted into an appropriate expression vector and appropriate host cells are transformed with the recombinant vector.
  • the cells are subjected to an appropriate screening process to identify recombinant cells producing the carrier polypeptide of interest. For example, lysates of samples of the cultures may be subjected to gel electrophoresis to identify those producing a polypeptide of the size expected for the carrier polypeptide. Further analysis may involve one of the above-described drug-binding assays.
  • a recombinant microbial strain producing the desired carrier polypeptide is cultured on a larger scale to produce the carrier polypeptide as needed.
  • composition comprising Polymeric Carrier and Drug
  • a single- or multi-domain version of the polymeric carrier derived from RBP is reacted with doxorubicin and/or other anthracyclines as described in Example 1. Unbound drug is removed by gel filtration. Carrier-bound drug is then lyophilized with a typical additive such as lactose. Upon reconstitution to form an aqueous solution, the composition is administered to patients with tumors. Improved tumor delivery and less cardiac toxicity (compared to administration of the free drug) are expected to be achieved, thereby allowing higher dose levels as well as administration to patients who are no longer eligible for treatment with adriamycin because of cumulative cardiac toxicity. The slow release of the drug from the polymeric carrier also maintains higher serum concentrations for longer periods of time.
  • a polymeric carrier such as one produced as described in Example 1 or 2 is covalently bound to streptavidin as follows. This is an alternative procedure to the methods for forming conjugates described in Example 1.
  • the SMCC-derivatized streptavidin is employed to form the desired product in the following reaction scheme: ##STR1## where the dotted line indicates non-covalent association between the drug and the drug binding domain and where y ranges from 1 to about 2, x ranges from about 5 to about 125, wherein R is the side chain of an amino acid n ranges from 1 to about 20, and n' ranges from about 1 to about 20 with n' being less than or equal to n.
  • the amino acid residues delimited by x constitute individual drug binding domains that non-covalently associate with the drug molecules as well as additional amino acid residues, if any, included for synthetic convenience or other purposes. There are n' drug binding domains which bind n drugs.
  • the binding of streptavidin to the proteinaceous polymeric carrier takes place via a free lysine residue on the polymeric carrier.
  • Example 1 The preparation of a polymeric carrier and the binding thereto to adriamycin, for example, has been described in Example 1, with the solubility of adriamycin potentially limiting the number of drug molecules that may be bound to this embodiment of the polymeric carrier of the present invention.
  • the remaining lysine residues on the drug bound polymeric carrier are derivatized with iminothiolane (Traut's reagent available from Pierce Chemical Company) to produce free thiols on the drug bound polymeric carrier. If cysteine residues are available for binding, derivatization of the lysine residues is unnecessary.
  • the thiols are then conjugated to the SMCC-derivatized streptavidin under suitable conditions to form a thioether linkage between the polymer and streptavidin.
  • the resulting immunoconjugate is mixed with the drug in a buffered solution, whereby the drug becomes associated with the polymeric carrier.
  • Non-bound drug is removed by gel filtration or dialysis.
  • the thus-produced conjugate of the present invention may be administered to a patient bearing a pretargeted biotin site to which streptavidin binds.
  • Drugs are associated with the polymeric carrier by a non-covalent interaction of the drug to the binding domain of the polymeric carrier having an affinity for that drug, as described in earlier examples.
  • lysine residues are present on the polymeric carrier and are available for binding to ligand such as biotin, the biotin-polymeric carrier-drug conjugate is formed in one step as shown below.
  • the drug bound polymeric carrier is reacted with biotin-NHS ester (available from Sigma Chemical Company) at basic pH to form the product conjugate.
  • the resulting immunoconjugate is mixed with the drug in a buffered solution, whereby the drug becomes associated with the polymeric carrier.
  • Non-bound drug is removed by gel filtration or dialysis.
  • the thus-produced conjugate of the present invention may be administered to a patient bearing a pretargeted avidin or streptavidin site to which biotin binds.
  • Biotinylated NR-LU-10 was prepared according to either of the following procedures. The first procedure involved derivatization of antibody via lysine ⁇ -amino groups. NR-LU-10 was radioiodinated at tyrosines using chloramine T and either 125 I or 131 I sodium iodide. The radioiodinated antibody (5-10 mg/ml) was then biotinylated using biotinamido caproate NHS ester in carbonate buffer, pH 8.5, containing 5% DMSO, according to the scheme below. ##STR3##
  • biotinylation was examined. As the molar offering of biotin:antibody increased from 5:1 to 40:1, biotin incorporation increased as expected (measured using the HABA assay and pronase-digested product) (Table 1, below). Percent of biotinylated antibody immunoreactivity as compared to native antibody was assessed in a limiting antigen ELISA assay. The immunoreactivity percentage dropped below 70% at a measured derivatization of 11.1:1; however, at this level of derivatization, no decrease was observed in antigen-positive cell binding (performed with LS-180 tumor cells at antigen excess). Subsequent experiments used antibody derivitized at a biotin:antibody ratio of 10:1.
  • NR-LU-10 was biotinylated using thiol groups generated by reduction of cystines. Derivatization of thiol groups was hypothesized to be less compromising to antibody immunoreactivity.
  • NR-LU-10 was radioiodinated using p-aryltin phenylate NHS ester (PIP-NHS) and either 125 I or 131 I sodium iodide. Radioiodinated NR-LU-10 was incubated with 25 mM dithiothreitol and purified using size exclusion chromatography.
  • the reduced antibody (containing free thiol groups) was then reacted with a 10- to 100-fold molar excess of N-iodoacetyl-n'-biotinyl hexylene diamine in phosphate-buffered saline (PBS), pH 7.5, containing 5% DMSO (v/v).
  • PBS phosphate-buffered saline
  • biotinylated antibodies (“antibody (lysine)" and "antibody (thiol)", respectively) were compared.
  • Molecular sizing on size exclusion FPLC demonstrated that both biotinylation protocols yielded monomolecular (monomeric) IgGs.
  • Biotinylated antibody (lysine) had an apparent molecular weight of 160 kD, while biotinylated antibody (thiol) had an apparent molecular weight of 180 kD.
  • Reduction of endogenous sulfhydryls to thiol groups, followed by conjugation with biotin may produce a somewhat unfolded macromolecule. If so, the antibody (thiol) may display a larger hydrodynamic radius and exhibit an apparent increase in molecular weight by chromatographic analysis.
  • Both biotinylated antibody species exhibited 98% specific binding to immobilized avidin-agarose.
  • biotinylated antibody species were either radiolabeled or unlabeled and were combined with either radiolabeled or unlabeled avidin or streptavidin. Samples were not boiled prior to SDS-PAGE analysis.
  • the native antibody and biotinylated antibody (lysine) showed similar migrations; the biotinylated antibody (thiol) produced two species in the 50-75 kD range. These species may represent two thiol-capped species. Under these SDS-PAGE conditions, radiolabeled streptavidin migrates as a 60 kD tetramer.
  • Radioiodinated biotinylated NR-LU-10 (lysine or thiol) was intravenously administered to non-tumored nude mice at a dose of 100 ⁇ g.
  • mice were intravenously injected with either saline or 400 ⁇ g of avidin.
  • saline administration blood clearances for both biotinylated antibody species were biphasic and similar to the clearance of native NR-LU-10 antibody.
  • biotinylated antibody lysine
  • avidin administration (10:1 or 25:1) reduced the circulating antibody level to about 35% of injected dose after two hours.
  • Residual radiolabeled antibody activity in the circulation after avidin administration was examined in vitro using immobilized biotin. This analysis revealed that 85% of the biotinylated antibody was complexed with avidin.
  • biotinylated antibody lysine 2 h post-avidin or post-saline administration were performed. Avidin administration significantly reduced the level of biotinylated antibody in the blood, and increased the level of biotinylated antibody in the liver and spleen. Kidney levels of biotinylated antibody were similar.
  • Certain antibodies have available for reaction endogenous sulfhydryl groups. If the antibody to be biotinylated contains endogenous sulfhydryl groups, such antibody is reacted with N-iodoacetyl-n'-biotinyl hexylene diamine.
  • one or more sulfhydryl groups are attached to a targeting moiety through the use of chemical compounds or linkers that contain a terminal sulfhydryl group.
  • An exemplary compound for this purpose is iminothiolane. As with endogenous sulfhydryl groups (discussed above), the detrimental effects of reducing agents on antibody are thereby avoided.
  • the monoclonal antibody-biotin conjugate is dissolved in PBS.
  • the pH of the solution is adjusted to 8.5 by addition of 0.5 M borate buffer, pH 8.5.
  • a DMSO solution of SMCC is prepared, and this solution is added dropwise to the vortexing protein solution. After 30 minutes of stirring, the solution is purified by G-25 (PD-10, Pharmacia, Piscataway, New Jersey) column chromatography to remove unreacted or hydrolyzed SMCC.
  • G-25 PD-10, Pharmacia, Piscataway, New Jersey
  • Lysine residues on the polymeric carrier are derivatized with iminothiolane (Traut's reagent available from Pierce Chemical Company) to produce free thiols on the polymeric carrier. If cysteine residues are available for binding, derivatization of the lysine residues is unnecessary.
  • the thiols are then conjugated to the SMCC-derivatized monoclonal antibody-biotin conjugate under suitable conditions to form a thioether linkage between the polymer and the monoclonal antibody.
  • the resulting immunoconjugate is mixed with the drug in a buffered solution, whereby the drug becomes associated with the polymeric carrier.
  • Nonbound drug is removed by gel filtration or dialysis.
  • the thus-produced conjugate of the present invention may be administered to a patient bearing a target site to which the antibody binds.
  • a patient presents with ovarian cancer.
  • a monoclonal antibody (MAb) directed to an ovarian cancer cell antigen is conjugated to biotin to form a MAb-biotin conjugate.
  • the MAb-biotin conjugate is administered to the patient in an amount in excess of the maximum tolerated dose of conjugate administrable in a targeted, direct label protocol (e.g., administration of monoclonal antibody-chelate-radionuclide conjugate) and is permitted to localize to target cancer cells for 24-48 hours.
  • an amount of avidin sufficient to clear nontargeted MAb-biotin conjugate and to bind to the targeted biotin is administered.
  • a biotin-polymeric carrier-drug conjugate of the type discussed in Example 7 above is dispersed in a pharmaceutically acceptable diluent and administered to the patient in a therapeutically effective dose.
  • the biotin-polymeric carrier-drug conjugate localizes to the targeted MAb-biotin-avidin moiety or is removed from the patient via the renal pathway.
  • a patient presents with colon cancer.
  • a monoclonal antibody (MAb) directed to a colon cancer cell antigen is conjugated to biotin to form a MAb-biotin conjugate.
  • the MAb-biotin conjugate is administered to the patient in an amount in excess of the maximum tolerated dose of conjugate administrable in a targeted, direct label protocol (e.g., administration of monoclonal antibody-chelate-radionuclide conjugate) and is permitted to localize to target cancer cells for 24-48 hours.
  • a streptavidin-polymeric carrier-drug conjugate of the type discussed in Example 6 above is dispersed in a pharmaceutically acceptable diluent and administered to the patient in a therapeutically effective dose. The streptavidin-polymeric carrier-drug conjugate localizes to the targeted MAb-biotin moiety or is removed from the patient via the patient's excretory system.
  • a patient presents with colon cancer.
  • a monoclonal antibody (MAb) directed to a colon cancer cell antigen is conjugated to biotin and to a polymeric carrier.
  • Drug is then non-covalently associated with the polymeric carrier portion of the conjugate to form a drug-polymeric carrier-MAb-biotin conjugate as described in Example 8 above.
  • the drug-polymeric carrier-MAb-biotin conjugate is administered to the patient in an amount approaching the maximum tolerated dose of conjugate administrable in a targeted, direct label protocol (e.g., administration of monoclonal antibody-chelate-radionuclide conjugate) and is permitted to localize to target cancer cells for 24-48 hours.
  • Streptavidin is dispersed in a pharmaceutically acceptable diluent and administered to the patient in a dose effective to cross-link the biotins that are now associated with the target site.
  • the streptavidin localizes to the targeted drug-polymeric carrier-MAb-biotin moiety conjugates and cross-links those conjugates, thereby inducing internalization thereof.

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US5871710A (en) * 1992-09-04 1999-02-16 The General Hospital Corporation Graft co-polymer adducts of platinum (II) compounds
US5607659A (en) * 1993-02-02 1997-03-04 Neorx Corporation Directed biodistribution of radiolabelled biotin using carbohydrate polymers
US5482698A (en) * 1993-04-22 1996-01-09 Immunomedics, Inc. Detection and therapy of lesions with biotin/avidin polymer conjugates
USRE39220E1 (en) 1994-05-11 2006-08-01 Genetic Applications, Llc Peptide-mediated gene transfer
WO1996000079A1 (fr) * 1994-06-27 1996-01-04 The General Hospital Corporation Produits d'addition copolymeres greffes de composes de platine (ii)
WO1996039183A1 (fr) * 1995-05-31 1996-12-12 Fred Hutchinson Cancer Research Center Compositions et procedes pour la liberation ciblee de molecules effectrices
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WO1997041898A1 (fr) * 1996-05-03 1997-11-13 Immunomedics, Inc. Immunotherapie-cible associee contre le cancer
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